Amplification of biological targets via on-chip culture for biosensing

ABSTRACT

The present invention, in part, relates to methods and apparatuses for on-chip amplification and/or detection of various targets, including biological targets and any amplifiable targets. In some examples, the microculture apparatus includes a single-use, normally-closed fluidic valve that is initially maintained in the closed position by a valve element bonded to an adhesive coating. The valve is opened using a magnetic force. The valve element includes a magnetic material or metal. Such apparatuses and methods are useful for in-field or real-time detection of targets, especially in limited resource settings.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of prior U.S. application Ser. No.14/157,378, filed Jan. 16, 2014, which in turn claims the benefit ofU.S. Provisional Application Nos. 61/857,029, filed Jul. 22, 2013, and61/870,841, filed Aug. 28, 2013, each of which is hereby incorporated byreference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under Contract No.DE-NA0003525 awarded by the United States Department of Energy/NationalNuclear Security Administration. The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Miniaturized systems can be useful for detecting various analytes,contaminants, and toxins. In particular, such systems can allow forreal-time or in-field detection of a target, while minimizing sample andreagent consumption. Point-of-care testing has wide applicability, notonly in diagnostic fields, but also in detecting pathogens or geneticmodifications in agricultural settings, as well as assessing biologicaland chemical threats in environmental screenings.

Despite significant progress in the development of biosensortechnologies, the utility of many assays remains limited. Generally,this is due to the inability of these assays to detect the biologicaltarget at or below the infectious dose (commonly 10²-10³ cells orspores). Also, some sensors lack the sensitivity and/or specificityrequired for detection of the desired target.

For use in remote or low resource settings, simpler sensor systems aredesired. For instance, such systems can include simplified modes ofoperation, reduced power use, and low production costs. When toxicagents are being detected, another benefit includes single-use systemsthat could be safely disposed. Accordingly, more components to implementsuch systems are needed.

SUMMARY OF THE INVENTION

The present invention features apparatuses having a magneticallyactuated valve and methods of their use. One example embodiment of theinvention is a single-use, normally-closed fluidic valve, which isopened using a magnetic force. The valve includes a valve seat(including an opening) and a valve element, which closes the opening ofthe valve seat.

In one example, the valve seat is made from a thin laminate(approximately 0.5 to 1.0 mm thick) that separates two chambers (e.g.,fluidic chambers) and is coated with adhesive. A hole cut into thelaminate serves as a fluidic via between the chambers, which istemporarily sealed closed with a valve element affixed over the via andadhered to the adhesive of the laminate.

The valve element is composed of a material that responds to an appliedmagnetic field. For instance, such materials include a magnetic materialor a metal. In one non-limiting use, the valve is opened by bringing anexternal magnet in proximity to the valve to provide a magnetic forcethat delaminates the valve from the adhesive coating. The externalmagnet is then physically moved to a location away from the via, whichremoves the valve opening the via for fluid flow between the chambers.In another embodiment, an integrated magnet is configured over the valveto provide an applied magnetic field to the valve element. In use, thisintegrated magnet is activated (e.g., by providing current to anintegrated electromagnet) to apply the magnetic field, thereby openingthe valve. To close the valve, the integrated magnet is deactivated(e.g., by removing current to the integrated electromagnet), therebyreleasing the valve element.

In particular, the present device can provide enhanced detection of oneor more targets. In particular, the device can be adapted to include oneor more chambers or reservoirs that allow for amplification of thetarget. For instance, if the target is a bacterium, then the device caninclude an incubation reservoir or chamber for increasing theconcentration of the bacteria in the test sample. In a similar manner,the concentration of any amplifiable target (e.g., a virus, a fungus, ora parasite) can be increased, as compared to that in the test sample. Inthis manner, amplification of the biological target prior to downstreambiodetection can improve the detection limit, allowing existingbiosensing technologies to effectively detect practical concentrationsor levels of the biological target. Accordingly, also described hereinare robust portable devices for amplification of biological targetsfacilitating subsequent biodetection.

Furthermore, such devices can be ultra-low cost, require no power orinstrumentation to operate, and can be operated by individuals withlittle to no technical training. Exemplary self-contained credit-cardsized devices can employ on-chip microculture methods to amplifybacteria prior to downstream detection, improving detection limits bymore than four orders of magnitude, where detection from a 10² spores/mLinitial inoculum is demonstrated herein.

In one aspect, the invention features an apparatus (e.g., a fluidic,microculture, and/or multilevel apparatus) including a first reservoirincluding at least one first outlet; a first valve element; a firstvalve seat conformed to support the first valve element in a positionthat closes the first outlet; and a layer of adhesive deposited on atleast a portion of the first valve seat such that the adhesive layer iseffective to releasably bond to a surface of the first valve elementwhen the first valve element is seated in the outlet-closed position. Insome embodiments, the first valve element is responsive to an appliedmagnetic field (e.g., by an external source and/or an integrated source,such as any described herein) of sufficient strength by detaching fromthe first valve seat and undergoing a displacement that causes the firstoutlet to open.

In some embodiments, the reservoir (e.g., first, second, third, or otherreservoir) has a branch conformed to receive the valve element (e.g.,first, second, third, or other valve element) in a location laterallydisplaced from the valve seat (e.g., the corresponding first, second,third, or other valve seat).

In some embodiments, the first outlet leads to a receiving chamber(e.g., a reaction chamber, a sample chamber, an incubation chamber, areagent chamber, a sterilization chamber, an assay chamber, and/or awaste chamber).

In other embodiments, the reservoir (e.g., first, second, third, orother reservoir) further includes at least one inlet (e.g., a chamber ora channel in fluidic communication with a sample port, a receivingchamber, a further channel).

In further embodiments, the apparatus includes a second reservoirincluding at least one (e.g., one, two, three, four, five, six, seven,eight, nine, ten, or more) second outlet(s); a second valve element; anda second valve seat conformed to support the second valve element in aposition that closes at least one second outlet. In some embodiments,the second valve element is responsive to an applied magnetic field(e.g., by an external or integrated source) of sufficient strength bydetaching from the second valve seat and undergoing a displacement thatcauses at least one second outlet to open.

In some embodiment, the apparatus includes a second layer of adhesivedeposited on at least a portion of the second valve seat such that thesecond adhesive layer is effective to releasably bond to a surface ofthe second valve element when the second valve element is seated in theoutlet-closed position.

In some embodiments, the first outlet leads to a third reservoir and thesecond outlet leads to the first reservoir; the first outlet leads tothe second reservoir and the second outlet leads to a third reservoir;or both the first and second outlets lead to the third reservoir.

In another aspect, the invention features an apparatus (e.g., a fluidic,microculture, and/or multilevel apparatus) including: a first reservoir(e.g., in a first level) including at least one first outlet; a secondreservoir (e.g., in a second level, where the second level is below thefirst level), where the first outlet is configured for fluidiccommunication between the first and second reservoirs; a first valveelement; a first valve seat conformed to support the first valve elementin a position that closes the first outlet; and a first layer ofadhesive deposited on at least a portion of the first valve seat andconfigured to releasably bond to a surface of the first valve elementwhen seated in the outlet-closed position. In some embodiments, thefirst valve element is responsive to an applied magnetic field ofsufficient strength by detaching from the first valve seat andundergoing a displacement that causes the first outlet to open. In otherembodiments, the apparatus is a microculture apparatus and furtherincludes a cell media (e.g., a stabilized cell media optionallyincluding one or more host cells) in the first reservoir.

In some embodiments, the apparatus further includes a third reservoir(e.g., in the second level) including a second outlet configured forfluidic communication between the first and third reservoirs; a secondvalve element; and a second valve seat conformed to support the secondvalve element in a position that closes the second outlet. In furtherembodiments, the second valve element is responsive to an appliedmagnetic field (e.g., by an external and/or integrated source) ofsufficient strength by detaching from the second valve seat andundergoing a displacement that causes the second outlet to open. Inother embodiments, the apparatus is a microculture apparatus and furtherincludes a sterilization agent (e.g., a stabilized sterilization agent)in the third reservoir.

In some embodiments, the apparatus includes a first and/or second layerof adhesive that is deposited on at least a portion of the second valveseat and configured to releasably bond to a surface of the second valveelement when seated in the outlet-closed position.

In yet another aspect, the invention features a method for operating adevice (e.g., a fluidic device or any apparatus described herein), themethod including applying a first magnetic field to a first valveelement adhesively bonded to a first valve seat so as to produce amagnetic force configured to detach the first valve element from thefirst valve seat; and by application of the first magnetic field,breaking the adhesive bond between the first valve element and the firstvalve seat, thereby causing displacement of the first valve element thatopens a first outlet from a first reservoir.

In some embodiments, the method includes, by opening the first outlet,causing a first substance (e.g., sample, reagent, gas, liquid,semi-solid, or solid) to move (e.g., flow) from the first reservoir intoa chamber, where the first outlet is configured to provide fluidiccommunication between the first reservoir and the chamber. In particularembodiments, the method further includes shaking and/or tapping thedevice or apparatus.

In other embodiments, the method includes applying a second magneticfield to a second valve element adhesively bonded to a second valve seatso as to produce a magnetic force configured to detach the second valveelement from the second valve seat; and by application of the secondmagnetic field, breaking the adhesive bond between the second valveelement and the second valve seat, thereby causing displacement of thesecond valve element that opens a second outlet from a second reservoir.In some embodiments, the first and second magnetic fields are the samemagnetic field. In other embodiments, the first and second magneticfields are different magnetic fields.

In some embodiments, the method includes by opening the second outlet,causing a second substance (e.g., sample, reagent, gas, liquid,semi-solid, or solid) to move (e.g., flow) from the second reservoirinto the first reservoir and/or the chamber; and/or causing the firstliquid to flow from the first reservoir and/or the chamber into thesecond reservoir. In particular embodiments, the method further includesshaking and/or tapping the device or apparatus.

In another aspect, the invention features a method for amplifying and/ordetecting a target in a sample, the method including: introducing thesample within a first reservoir of a first apparatus, where the firstapparatus includes a first outlet in fluidic communication with thefirst reservoir (e.g., where the first apparatus is configured to allowfor in-field or real-time detection of the target); and incubating thesample within the first reservoir, thereby amplifying the target withinthe sample and providing an amplified sample. In some embodiments, thesample includes an amplifiable target (e.g., a bacterium, a virus, aparasite, a protozoon, a helminth, or a fungus).

In some embodiments, the method includes opening the first outlet (e.g.,after the introducing step, before the incubating step, or after theincubating step) by applying a magnetic field to a first valve elementadhesively bonded to a first valve seat so as to produce a magneticforce configured to detach the first valve element from the first valveseat.

In another embodiment, the method further includes introducing theamplified sample within an assay chamber, where the first outlet leadsto the assay chamber and the assay chamber is configured to include oneor more detection agents (e.g., a dye, a particle, a marker, or a label,or any agent described herein) for identifying the target, therebyidentifying whether or not the target is present within the sample. Inparticular embodiments, the method further includes shaking and/ortapping the device or apparatus.

In some embodiments, the assay chamber is provided in the firstapparatus or in a second apparatus having an inlet in fluidiccommunication with the assay chamber (e.g., where the inlet is influidic communication with the first outlet and/or the second outlet inthe first apparatus).

In some embodiments, the method further includes sterilizing theamplified sample during or after the incubation step, and/or afteridentifying the target. In further embodiments, the sterilizing stepincludes opening a second outlet in fluidic communication between asecond reservoir and the assay chamber, by applying a magnetic field toa second valve element adhesively bonded to a second valve seatconformed to support the second valve element in a position that closesthe second outlet, where the magnetic field produces a magnetic forceconfigured to detach the second valve element from the second valveseat; and introducing a sterilization agent from the second reservoirinto the assay chamber. In particular embodiments, the method furtherincludes shaking and/or tapping the device or apparatus. In someembodiments, the fluidic communication between the second reservoir andthe assay chamber occurs through an open passageway in the firstreservoir.

In some embodiments, the method further includes measuring the presenceof a detectable signal from the detection agent, e.g., byelectrochemical, colorimetric, fluorescent, western blot,immunohistochemistry, immunoassay, immunochromatography, radioimmunoassay, optical immunoassay, enzyme immunoassay, chemiluminescence,and/or electrochemiluminescence methods. In other embodiments, themeasuring step includes detection by lateral flow assay and/or detectionof a plurality of targets (e.g., one or more bacteria, viruses,parasites, protozoa, helminths, fungi, food-borne pathogens, weaponizedpathogens, or any target described herein).

In any of the embodiments, the valve element (e.g., first, second,third, or any valve element) includes a magnetic material (e.g., apermanent magnet, a neodymium magnet, a samarium-cobalt magnet, aferrite magnet, and/or an alnico magnet) and/or a metal (e.g., iron,nickel, cobalt, gadolinium, neodymium, samarium, steel, magnetite, aferrite, as well other metals, alloys, or composites capable of beingmagnetized). In any of the embodiments, the first, second, third, or anyvalve element is a disk.

In any of the embodiments, the first, second, third, or any valveelement is releasably bonded to a first valve seat by an adhesive layeror a portion thereof.

In any of the embodiments, two or more valve elements (e.g., first andsecond valve elements, first and third valve elements, second and thirdvalve elements, etc.) actuate in the same direction or in differentdirections.

In any of the embodiments, the reservoir (e.g., first, second, third, orother reservoir) is a reaction chamber, a sample chamber, an incubationchamber, a reagent chamber, a sterilization chamber, an assay chamber,or a waste chamber.

In any of the embodiments, the reservoir (e.g., first, second, third, orother reservoir) includes, independently, one or more sterilizationagents (e.g., stabilized sterilization agents, as described herein),detection agents (e.g., an electroactive or electrocatalytic detectionagent), labels (e.g., an electroactive or electrocatalytic detectionagent or label), amplifying agents, capture agents, cell media (e.g.,stabilized sterilization agents, as described herein, and optionallyincluding host cells), cells (e.g., living host cells for the target),detergents, surfactants, buffers, alcohols, preservatives, blockingagents, beads, or combinations thereof.

In any of the embodiments, the reservoir (e.g., first, second, third, orother reservoir) includes a sterilization agent (e.g., any describedherein, including a stabilized sterilization agent or a sterilizationagent in solid, semi-solid, liquid, or gas form).

In any of the embodiments, the reservoir (e.g., first, second, third, orother reservoir) includes an inlet for introducing a sample, and theapparatus is completely sealed except for the inlet.

In any of the embodiments, the reservoir (e.g., first, second, third, orother reservoir) includes a cell medium (e.g., any described herein,including a stabilized cell medium, optionally including living hostcells for the target).

In any of the embodiments, the reservoir (e.g., first, second, third, orother reservoir) is further conformed to contain a capillary bed forlateral flow assay.

In any of the embodiments, the apparatus, device, or method includes anintegrated source (e.g., an integrated permanent magnet, a magnetic coil(e.g., a solenoid), a metallic element to attract the valve element, oran electromagnet) configured to provide the applied magnetic field tothe valve element (e.g., first, second, third, or other valve element).

In any of the embodiments, the apparatus, device, or method isconfigured for detection of one, two, three, four, five, six, seven,eight, nine, ten, or more targets (e.g., any target described herein).

In any of the embodiments, the apparatus, device, or method includesin-field amplification and/or detection of one or more targets (e.g.,amplification and/or detection of one or more targets in less than about24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour,30 minutes, 20 minutes, or less, and optionally configured to use no orminimal power for detecting the target).

In any of the embodiments, the apparatus, device, or method includesreal-time amplification and/or detection of one or more targets (e.g.,amplification and/or detection of one or more targets in less than about2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10minutes, 5 minutes or less, and optionally configured to use no orminimal power for detecting the target).

In any of the embodiments, the apparatus, device, or method isconfigured to allow for amplification and/or detection in a resourcelimited environment (e.g., an environment having limited access to a laband/or trained staff). In one non-limiting embodiment, the deviceincludes one or more additional components (e.g., any described herein).In another non-limiting embodiment, the device includes one or morecomponents selected from the group of a separation/extraction component,a heating component, a pump, a membrane, a multifunctional sensor, alight-emitting diode, an active circuit element, a passive circuitelement, a power source, a photodiode, and a telemetry unit.

In any of the embodiments, the method includes use of any device orapparatus (e.g., fluidic, microculture, and/or multilevel apparatus)described herein.

DEFINITIONS

By “about” is meant +/−10% of the recited value.

By “fluidic communication,” as used herein, refers to any duct, channel,tube, pipe, chamber, or pathway through which a substance, such as aliquid, gas, or solid may pass substantially unrestricted when thepathway is open. When the pathway is closed, the substance issubstantially restricted from passing through. Typically, limiteddiffusion of a substance through the material of a plate, base, and/or asubstrate, which may or may not occur depending on the compositions ofthe substance and materials, does not constitute fluidic communication.

By “microfluidic” or “micro” is meant having at least one dimension thatis less than 1 mm. For instance, a microfluidic structure (e.g., anystructure described herein) can have a length, width, height,cross-sectional dimension, circumference, radius (e.g., external orinternal radius), or diameter that is less than 1 mm.

As used herein, the terms “top,” “bottom,” “upper,” “lower,” “above,”and “below” are used to provide a relative relationship betweenstructures. The use of these terms does not indicate or require that aparticular structure must be located at a particular location in theapparatus.

Other features and advantages of the invention will be apparent from thefollowing description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A provides a schematic, cross-sectional view of an apparatus 100embodying an aspect of the present invention, including two fluidicchambers 310, 610 that intercommunicate through a via 450 that issealable by a permanent magnet 800. FIG. 1B is a schematic of magneticvalving used to control fluidic access between regions of an exemplaryB. anthracis detection device.

FIG. 2A provides a further, partially schematic, view of an apparatus1000 embodying an aspect of the present invention. The view of FIG. 2Ais an isometric, exploded view of an apparatus 1000 constructed in sixlayers 1100-1600 and containing two valves 1450/1800, 1460/1810 andthree fluidic chambers 1310, 1510, 1520. FIG. 2B provides a perspective,exploded view of the various layers of the apparatus in FIG. 2A.

FIG. 3 provides top and bottom view photographs of an exemplary deviceprior to opening of the valves. The exemplar device includes a samplechamber 2310, a sterilization chamber 2520, a lateral flow assay (LFA)chamber 2510, a first valve (sample valve or “Valve 1”) 2450, and asecond valve (sterilization valve or “Valve 2”) 2460.

FIG. 4 provides top view photographs of an exemplary device afteropening the sample valve (“Valve 1”). Shown are the magnet of Valve 1(labeled with black arrow), the open via for this valve (indicated bywhite arrowhead), and the external magnet used to open the valve(labeled with black arrow).

FIG. 5 provides top view photographs of an exemplary device afteropening the sterilization valve (“Valve 2”). Shown are the magnet ofValve 2 (labeled with black arrow) and the open via for this valve(labeled with black arrow).

FIG. 6 provides elevational view photographs of an exemplary deviceafter mixing of the sterilization solution in the sample chamber 2310and the LFA chamber 2510. Shown in the sterilization chamber 2520 is themagnet of the sterilization valve (at lower right corner of thischamber). As can be seen, the sample chamber 2310 is designed to allowthe magnet of the sample valve to rest in a recess (at lower left cornerin the bottom photograph). In this manner, the magnet does not blockflow within the LFA chamber 2510.

FIG. 7 provides front view photographs of an exemplary device for LFA.

FIG. 8 provides side view photographs of an exemplary device with closedvalves (top photograph and background of bottom photograph) and withopen valves (foreground of bottom photograph).

FIG. 9A-9B provides a perspective, exploded view of the various layersthat were assembled to create an illustrative prototype 3000 embodyingan aspect of the present invention.

FIG. 10 provides a plan view of the respective layers in FIG. 9A-9B.

FIG. 11A-11B shows (A) a schematic and (B) a photograph of an exemplaryfully assembled B. anthracis detection device with on-chip microculturefor biological target amplification. In FIG. 11B, the on-chipmicroculture chamber is filled with dye. The entire device is about thesize of four stacked credit cards.

FIG. 12A-12B shows photographs of an exemplary B. anthracis detectiondevice in use.

FIG. 13A-13C shows the effect of culture length on a lateral flowimmunoassay (LFA) in exemplary B. anthracis detection devices. Theinitial spore count was an inoculation with 100 spores.

DETAILED DESCRIPTION OF THE INVENTION

An exemplary principle of operation is illustrated in FIG. 1A, whichprovides a schematic, cross-sectional view of an apparatus 100 includingtwo fluidic chambers 310, 610 that intercommunicate through a via 450that is sealable by a permanent magnet 800. As shown in the figure byway of illustration only and not by way of limitation, the apparatus isconstructed from six sheets, or laminae. The outer sheets 200, 700 aresupport structures. Each of the two sheets 300, 600 adjacent therespective outer sheets is perforated to provide fluidic chambers 310,610.

The fifth and sixth sheets 400, 500 form an adjacent pair that issituated between the two perforated sheets 300, 600. The fifth and sixthsheets 400, 500 are perforated to provide a via 450 between the twofluidic chambers 310, 610. The perforations in the fifth and sixthsheets are exemplarily circular in cross section and concentric, withthe lower perforation in sheet 500 of slightly smaller diameter than theupper perforation in sheet 400 so as to provide a shoulder 455 on whicha valve element 800 rests.

In one embodiment, the valve element 800 is a permanent magnet thatinterfaces with the via 450. As can be seen in this exemplary device,the valve element 800 is a cylindrical magnet in order to interface withcircular via 450. A skilled artisan would understand how to match thecross-sections of the valve element and the via to form a closed valve.

The upper surface of the shoulder 455 is coated with an adhesive layer.In the “valve closed” configuration, the valve element is seated on theshoulder 455 and temporarily bonded to the underlying layer by theadhesive (FIG. 1A, left). In that configuration, the valve element 800seals the via 450 (in an outlet-closed position).

To actuate the valve, i.e., to transform it to the “valve open”configuration, an external permanent magnet is brought into proximity tothe valve element (FIG. 1A, right). The force of magnetic attractionbetween the valve element 800 and the external magnet breaks theadhesive bond and lifts the valve element 800 off of its seat 455.Lateral motion of the external magnet then slides the valve element 800to a neutral location where it no longer seals the via 450 (e.g., arecess or a corner of the chamber). Of course, the use of an externalpermanent magnet is merely illustrative and can be replaced, forexample, by the electrical activation of magnetic coils or by using ametallic structure to which the valve element is attracted.

FIG. 1B provides an exemplary device for use with a lateral flow assay(LFA). The device includes a microculture chamber 315 and an LFA chamber615 having a capillary bed (shown as gray layer within chamber 615),where these two chambers are fluidically connected by an openable valveelement 805. The valve seat includes an adhesive layer 505, as describedherein.

A more realistic, but still partially schematic, illustration of theinvention is provided by FIG. 2A, which is an isometric, exploded viewof an apparatus 1000 constructed in six layers 1100-1600 and containingtwo valves 1450/1800, 1460/1810 and three fluidic chambers 1310, 1510,1520. As seen in the figure, the first (top) layer 1100 is a structurallayer suitable for bearing printed legends and the like, and perforatedwith a fill hole 1110 that is sealable by a plug 1120. The second layer1200 is likewise a structural layer perforated with a portion of thefill hole 1210. In the example illustrated, the plug 1120 has a widertop portion that seats in the perforation 1110 in the top layer 1100 anda narrower bottom portion that seats in the perforation 1210 in thesecond layer 1200.

The third layer 1300 is perforated to provide one of the fluidicchambers 1310, which in this example is a reservoir for a liquid sample,designated the sample chamber.

The fourth layer 1400 includes two vias 1450, 1460 and the seats for thetwo corresponding magnetic valve elements 1800, 1810. In someimplementations, the fourth layer can be a composite layer made, e.g.,from two, three, or more sheets, as described herein.

The fifth layer 1500 is perforated to provide the two further fluidicchambers 1510, 1520, whose bottoms are sealed by the sixth layer 1600,which is a structural layer. In this example, one of the fluidicchambers 1510 is designated as the Lateral Flow Assay (LFA) chamber, andthe other is designated as the sterilization chamber 1520. In oneexemplary embodiment, the LFA chamber is long and narrow (e.g., having alength to width ratio of 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1,12:1, or greater, as well as ranges therebetween). It is conformed forplacement therein of a capillary bed 1560, such as a porous paper strip,for performing diagnostic procedures by immunochromatography or thelike. Although the present invention is especially well suited for suchuses, it has a wide range of potential applications that are not limitedto the biomedical sphere. One of the two valves, designated the samplevalve 1450/1800, is positioned so that when actuated, it admits liquidthrough its via 1450 from the sample chamber 1310 to one end of the LFAchamber 1510. In operation, the admitted sample liquid is absorbed by aportion of the capillary bed 1560 proximal the via 1450. From there, thesample liquid propagates by capillary action toward the other end of thecapillary bed 1560.

As noted, the fifth layer 1500 is perforated to provide the LFA chamber1510 and one further fluidic chamber, which is here designated thesterilization chamber 1520. The sterilization chamber 1520 is meant tohold a quantity of bleach or other sterilizing agent. The valve1460/1810 for the sterilization chamber 1520 is aligned so that whenactuated, it will admit the sterilizing agent through its via 1460 intothe sample chamber 1310, then through the sample via 1450, from which itcan also eventually penetrate into the LFA chamber 1510.

FIG. 2B provides an exploded view showing that each of the six layers1100-1600 can include further additional substrates or laminae. Forinstance, first layer 1100 can include a film substrate 1101 includinglabels and a first structural substrate 1102. The second layer isprovided as a single substrate 1201. To laminate the first and secondlayers, an adhesive layer can be present either on the bottom of thefirst structural substrate 1102 or the top of the second substrate 1201.For any of the layers described herein, a skilled artisan would be ableto design and construct one or more adhesive layers in a useful mannerfor assembling the apparatus.

The third layer 1300 can include multiple substrates, including anadhesive film 1301, a polymer substrate 1302, and another adhesive film1303. As can be seen, each of these substrates can include an opening1311-1313 to define a reservoir or sample chamber 1310 in the apparatus.

The fourth layer 1400 can include multiple substrates 1401-1403, whichare designed to include valve seats for the magnetic valves. As can beseen, circular vias are provided in each of these substrates, where vias1451-1453 form a first valve seat, and vias 1461-1463 form a secondvalve seat. The first valve seat is designed so that the via 1451 in theupper substrate 1401 is slightly larger than the vias 1452, 1453 in thelower substrates 1402, 1403. In this design, the first valve elementwould rest within via 1451 and the shoulder would be provided by theedge of vias 1452, 1453. In contrast, the second valve seat is designedso that the second valve element would rest within a via 1463 in thelower substrate 1403. For instance, the vias 1461, 1462 in the uppersubstrates 1401, 1402 are slightly smaller than the via 1463 in thelower substrate 1403. In this design, the second valve element wouldrest within via 1463 and the shoulder would be provided by the edge ofvias 1461, 1462. Thus, the first valve element actuates into an openposition by moving towards the top of the device, whereas the secondvalve element actuates into an open position by moving towards thebottom of the device. In this manner, a set of valves can be designed toactuate in different directions.

In some embodiments, substrate 1402 is an adhesive film or includes sucha film, which provides an adhesive interface for both of the valves.

The fifth layer can include multiple substrates, including an adhesivefilm 1501, a structural substrate 1502, and another adhesive film 1503.As can be seen, adhesive film 1503 provides an exposed surface throughthe opening 1512, where this exposed surface can be useful for placing acapillary bed. These substrates can include an opening 1521-1523 todefine a reservoir or sterilization chamber 1520 in the apparatus, aswell as another opening 1511, 1512 to define the reaction chamber or LFAchamber 1510. Finally, the last layer 1600 can include a structuralsubstrate 1601.

In some implementations, it may be useful to conform the reservoirs withside branches into which the released valve element can be directed bylateral movement of the external or integrated magnetic source. Suchplacement of the valve element can be advantageous in order to preventit from subsequently interfering with flow through the vias. Exemplaryside branches can be included in the sample chamber (see, e.g., thelower left side of the sample chamber 1311 in FIG. 2B, which provides anelongated branch that extends past the reaction chamber 1511 when thedevice is laminated) and/or in the sterilization chamber (see, e.g., thelower right side of the sterilization chamber 1521 in FIG. 2B, whichprovides a constricted side branch to place the valve element afteropening the valve). In some other implementations, mutual repulsionbetween the respective valve elements may be sufficient to prevent themfrom blocking the vias, especially if the chamber thickness is too smallto permit the valve elements to flip over.

An operative prototype was produced that was about 2.75 inches long,about 1.875 inches wide, and about 0.331 inches thick. Furtherminiaturization is possible. Miniaturization will ultimately be limitedby the desired adhesion between the valve element and the valve seat,which for a given adhesive and given processing conditions will decreaseas the contact area decreases, and by the viscosities or granularity ofthe various fluids and agents, which will lead to greater flowresistance as the vias and reservoirs become more constricted. Thediminution of adhesive force can be compensated by increased time andpressure in the assembly process up to a saturation point beyond whichfurther increases will not be beneficial. The increase in flowresistance might, in some implementations, be compensated by activepumping, e.g., through manually operated plungers or bellows, or throughthe motion of a massive free-body ram driven by shaking the device.

FIGS. 3-8 are based on photographs of prototype devices of the kindillustrated in FIG. 2A-2B in various stages of operation. The samplechamber 2310 is initially filled with an aqueous solution of blue foodcoloring, visible as the gray liquid in the figures. The sterilizationchamber 2520 is initially filled with an aqueous bleach solution, whichupon intermixing will decolorize the dyed solution.

FIG. 3 provides top and bottom views of a prototype device denominated“Sample 1” before any valves 2450, 2460 are opened. FIG. 4 provides topviews of the same device, at different levels of magnification, afterthe sample valve has been opened and liquid has begun to flow into theLFA chamber 2510. FIG. 5 provides top views of the same device, atdifferent levels of magnification, after the sterilization valve hasbeen opened and bleach has begun to flow from the sterilization chamber2520 into the sample chamber 2310 and LFA chamber 2510. FIG. 6 providesan elevational view (top side facing the camera) of the same device,stood on an edge, after substantially complete intermixing of the bleachsolution with the contents of the sample 2310 and LFA 2510 chambers.

FIG. 7 provides a view, similar to the view of FIG. 6, of a secondprototype device, denominated “Sample 2”, prior to opening any valves.As indicated in the figure, the sample chamber contains 0.3 mL of dyedwater, the sterilization chamber contains 1.6 mL of a 5% bleachsolution, and the LFA chamber is empty. FIG. 8 provides in the top imageand in the background of the bottom image a view of Sample 2 similar tothe view of FIG. 7. FIG. 8 also provides for comparison, in theforeground of the bottom image, a view of Sample 1 similar to the viewof FIG. 6.

In another example, the device is designed to accommodate a microculturechamber for LFA analysis. As shown in FIG. 9A, the device 3000 caninclude seven layers 3100-3700 and contain two valves 3800/3450,3810/3460 and three fluidic chambers 3310, 3610, 3620. The device canfurther include a plug 3120, a seal 3125, and a fill hole 3110. Thechambers can include a microculture chamber 3310, a sterilizationchamber 3620, and an LFA chamber 3610 for placement of an LFA capillarybed 3660.

As seen in FIG. 9B, each of the layers in FIG. 9A can include furthersubstrates. FIG. 10 shows a plan view of these substrates. The firstlayer 3100 can include a structural substrate 3101 having recessedregions 3150, 3151 for placement of an external magnet and a label 3150.Recessed regions 3150, 3151 are aligned with the valve elements, suchthat placing the external magnet in recess 3150 will align this externalmagnet with the first valve element. Similarly, placement of theexternal magnet in recess 3151 will result in alignment with the secondvalve element. The second layer 3200 includes a structural substrate3201. The third layer 3300 includes an adhesive film 3301, a structuralsubstrate 3302, and another adhesive film 3303, where each has anopening 3311-3313 for the microculture chamber 3310.

The fourth layer 3400 includes a structural substrate 3401 having vias3451, 3461, and the fifth layer 3500 includes a structural substratehaving an adhesive film 3501 and vias 3452, 3462. The first valve isformed from vias 3451, 3452, where the via 3451 in the upper substrate3401 is slightly larger than the via 3452 in the lower substrate 3501.The second valve is formed from vias 3461, 3462. Similar to the firstvalve, the second valve is designed having the via 3461 in the uppersubstrate 3401 to be slightly larger than the via 3462 in the lowersubstrate 3501. As both valves have a larger via in the upper substrate,both valves are actuated in the same direction.

The sixth layer 3600 includes an adhesive film 3601 and a structuralsubstrate 3602, and the seventh layer 3700 includes an adhesive film3701 and a structural substrate 3702. As can be seen, some of theselayers include an opening for the sterilization chamber 3621-3623 andthe LFA chamber 3611, 3612.

Valves

The present apparatus can include one or more valves to interconnectclosed or open structures. The valves include at least a valve element,a valve seat disposed within the device, and an adhesive interfaceprovided between a surface of the valve element and a surface of thevalve seat. In particular, the valve includes two structural componentsto ensure a closed valve: (i) an adhesive bond between the adhesiveinterface with the valve element and valve seat and (ii) matching ornear matching of the cross-sections between the valve element and valveseat. Furthermore, the valves can be designed to actuate in differentdirections (e.g., as described for the fourth layer in FIG. 2B) or inthe same direction (e.g., as described for the valves in FIG. 10).

The valves can be positioned to interconnect two or more closed or openstructures. Exemplary structures include a chamber or reservoir, achannel, a fill-hole, or a well. In some embodiments, the structure canbe closed (i.e., completely enclosed by surrounding walls and optionallyincluding one or more outlets, inlets, or vias for fluidic communicationwith other structures). In other embodiments, the structure can beopened, i.e., having one or more walls and also having one or more sidesthat open to the environment. In particular embodiments, the structureis a closed chamber or channel including one or more inlets, outlets, orvias, where each of the inlet, outlet, or via is connected to anotherclosed chamber or channel. In this embodiment, the valve can bepositioned to have a valve seat substantially surrounding the inlet,outlet, or via, such that the valve element sits within the valve seatand substantially blocks the inlet, outlet, or via in the closedposition.

The valve element can be formed from any useful material that isresponsive to an applied magnetic field, such as a magnetic material ora metal. Exemplary magnetic materials include a neodymium magnet (alsoknown as NdFeB), a samarium-cobalt magnet, a ferrite magnet, an alnicomagnet, or any other permanent magnets. Exemplary metals include iron,nickel, cobalt, gadolinium, neodymium, samarium, steel, magnetite, aferrite, as well other metals, alloys, or composites thereof, and anycapable of being magnetized. The valve seat can be formed from anyuseful material, e.g., such as any polymer described herein.

The adhesive interface can include any useful adhesive. Exemplaryadhesives include a pressure sensitive adhesive (e.g., an acrylic,silicon, or acrylic-hybrid based adhesive optionally including a supportlayer), an acrylic adhesive, an acrylic-hybrid adhesive, a siliconeadhesive, and/or an adhesion promoter (e.g., Dow Corning® 1200 primer,including light aliphatic petroleum solvent naptha, xylene, tetrapropylorthosilicate, tetrabutyl titanate, ethylene glycol methyl ether, tetra(2-methoxyethoxy) silane, and/or ethylebenzene).

The valve can be actuated by any useful applied magnetic field. Forinstance, FIG. 1A shows an external permanent magnet to actuate thevalve element by applying an external magnetic field, but other sourcesfor providing or inducing a magnetic field can be used. Exemplarysources include a permanent magnet, a magnetic coil (e.g., a solenoid),a metallic element to attract the valve element, and an electromagnet(e.g., an integrated electromagnet configured to be activatedelectronically).

The source providing the applied magnetic field can be an externalsource or a source that is integrated with the device. For instance andwithout limitation, an exemplary external source is provided in FIG. 1B,where the external magnet (or external source) applies a magnetic fieldto the valve (e.g., a thin disk magnetic valve 805 in this figure). Inanother instance, the source can be integrated with the device. Forexample and without limitation, the integrated source is provided withina layer of the device. In another embodiment, the integrated source isembedded between two or more layers within the device. Accordingly, anyof the sources described herein (e.g., a metallic element to attract thevalve element, an electromagnet, etc.) can be an external source or anintegrated source.

In some embodiments, the integrated source is an integratedelectromagnet configured to be activated electronically (e.g.,configured to include one or more electrical components, such as anelectrode, a power source, a wire, etc., where activation of theelectrical component induces current that activates the electromagnet).Such an integrated electromagnet can be configured in any useful manner(e.g., provided within a layer of the device or embedded between two ormore layers within the device, as well as positioned above the valveelement, thereby allowing the valve element to respond to the appliedmagnetic field from the activated electromagnet). Exemplary integratedelectromagnets include one or more magnetic particles (e.g., a thinmembrane or layer including a magnetic nanocomposite material or bead,such as a rare earth magnetic powder, including an Nd—Pr—Ce—Fe—B alloy(e.g., MQP-12-5 isotropic powder), an Nd—Fe—B alloy, an Nd—Pr—Fe—Balloy, an Nd—Co—Fe—B alloy, an Nd—Ce—Fe—B alloy, an Nd—Pr—La—Fe—B alloy,an Nd—Nb—Fe—B alloy, a Pr—Fe—B alloy, or a Pr—Co—Fe—B alloy), one ormore magnetic layers, or a solenoid electromagnet, where each canoptionally further include one or more electrical components to activatethe integrated electromagnet (e.g., one or more electrodes, such asmicropatterned electrodes; a wire; or a power source, such as an AC orDC power source) or one or more magnetic field concentrators.

Reservoirs and Chambers

The present apparatus can include one or more reservoirs or chambers,which can be designated for a particular use. The terms “reservoir” and“chamber” are used interchangeably.

Particular uses for such reservoirs and chambers include a samplechamber for receiving and/or storing a test sample, an incubationchamber for incubating a test sample (e.g., to amplify one or moretargets and optionally containing media and/or host cells for suchamplification), a reagent chamber containing one or more reagents fordetecting one or more targets, a sterilization chamber containing one ormore reagents to sterilize or disinfect the test sample (e.g.,containing one or more sterilization agents, as described herein), anassay chamber for conducting one or more assays to detect one or moretargets (e.g., an assay chamber containing a capillary bed for a lateralflow assay), and/or a waste chamber for storing one or more by-productsof the assay. Each of these chambers can be interconnected by a valve(e.g., including a valve element and a valve seat) and/or a channel thatcan optionally include a valve in its fluidic path.

Further, these chambers can be configured to perform particular reactionsteps. For instance, as shown in FIG. 11B, the device is labeled withnumbers “1” to “5” to indicate the particular reaction steps, and thechambers are designed to allow these steps to be conducted in theindicated order. The first step (indicated by “1” on the upper leftportion of the device in FIG. 11B) includes introducing the test sampleto the sample port 4110 located at the upper portion of the culturechamber 4310. Second (indicated by “2” in this figure), the sample isincubated in the culture chamber 4310 containing the media (see FIG.11A). Next (indicated by “3” in FIG. 11B), the LFA valve 4800/4450 isopened by applying an external magnet to the valve element (labeled “B”in FIG. 11A) and moving the valve element away from the via (in thedirection of the black arrow in FIG. 11A). In this particular device,valve element B actuates by moving towards the top side of the device.

Then (indicated by “4” in FIG. 11B), the amplified sample flows into theLFA 4660 chamber for biodetection. Finally (indicated by “5” in FIG.11B), the sterilization valve 4810/4460 is opened by applying anexternal magnet to the bottom side of the device, as this valve elementactuates by moving towards the bottom side of the device. Upon openingthis valve, bleach solution (or any sterilization agent, e.g., anydescribed herein) in the sterilization chamber 4620 moves or flows intothe culture chamber 4310 and the LFA 4660 chamber. The user mayoptionally shake or tap the device to facilitate mixing of the contents.In particular, if one or more agents are in solid form (e.g., astabilized sterilization agent in powdered form), then flow of theagents into the chambers may be assisted by shaking and/or tapping thedevice.

Optionally, the valve element for the sterilization valve can be placedin the position labeled “C” in the lower right portion of thesterilization chamber 4620 in FIG. 11A. In this position, the valveelement possesses limited movement and does not block the flow of thebleach solution throughout the chambers in the device. In a similarmanner, additional chambers and labels can be included to performparticular reactions in a particular order.

Multilevel Apparatus

The present apparatus can take any useful form, such as a multilevelapparatus having multiple layers. For instance, the apparatus can beconfigured to include one or more chambers, channels, and valves(including valve elements, valve seats, vias, and adhesive layers) inparticular layers. These layers can be designed to optimize themechanism of the valve, to accommodate multiple reaction steps in aplurality of chambers, to simplify production of the device, as well asany other design considerations.

The multilevel apparatus can include one or more components tofacilitate assembly or usage of the apparatus. For instance, adhesivelayers can be used between any structural layers to facilitatelaminating and assembling of the device. In another example, alignmentholes or markings can be used to position layers and openings in anappropriate manner. Such a device can include one or more labels todirect the user.

Furthermore, the multilevel apparatus can be designed to include valvesthat actuate in the same direction or in different directions. Forinstance, substrates 3401, 3501 in the device of FIG. 10 are designed tohave larger vias in the upper substrate 3401 and smaller vias in thelower substrate 3501. In this manner, both valves actuate in the samedirection (i.e., toward substrate 3103 or the top side of the assembledapparatus). As shown in FIG. 9A, both valve elements 3800, 3810 sit on avalve seat on the top side of the layer 3400.

In another embodiment, the valves actuate in different directions. Forinstance, as seen in FIG. 2B, substrates 1401-1403 are designed to havevalve seats on opposing sides of the layer, where the larger via 1451for the first valve is in the upper substrate 1401, but the larger via1463 for the second valve is in the lower substrate 1403. As shown inFIG. 2A, the first valve element 1800 sits on a valve seat on the topside of layer 1400, and the second valve element 1810 sits on a valveseat on the bottom side of the same layer 1400. In this manner, thevalves in a multilevel apparatus can be configured and operated in anyuseful way.

Multilevel or monolithic structures can be constructed using any usefulmethod. Exemplary methods of fabrication include rapid prototyping,microfabrication (e.g., by casting, injection molding, compressionmolding, embossing, ablation, thin-film deposition, and/or ComputerNumerically Controlled (CNC) micromachining), photolithography, etchingtechniques (e.g., wet chemical etching, reactive ion etching,inductively coupled plasma deep silicon etching, laser ablation, or airabrasion techniques), methods for integrating these structures intohigh-throughput analysis equipment (e.g., integration with a microplatereader or a control instrument, such as a computer), methods forfabricating and integrating valves (e.g., one or more pneumatic valves),methods for integrating structures with a transducer array, methods formodifying surfaces (e.g., by including a layer of extracellular matrixcomponents, such as fibronectin (FN), laminin, Matrigel™, and/or RGDpeptide), methods for including one or more capture arrays (e.g., acapture array including one or more capture agents provided in ahigh-density array on a substrate), and methods for providing vias orinlets (e.g., by piercing, drilling, ablating, or laser cutting), suchas those described in U.S. Pat. No. 8,257,964; and U.S. Pub. Nos.2012/0231976, 2012/0214189, 2011/0129850, 2009/0251155, and2009/0036324, each of which is incorporated herein by reference in itsentirety.

Materials

The present apparatus can be formed from any useful material. Exemplarymaterials include a polymer, such as polymethyl methacrylate (PMMA),polyethylene terephthalate (PET, e.g., biaxially-oriented PET orbo-PET), an acrylic polymer, poly(dimethylsiloxane) (PDMS),polycarbonate (PC), cyclo-olefin copolymer (COC), polyethyleneterephthalate glycol (PETG), polyethylene (PE, such as branchedhomo-polymer PE), polyvinylchloride (PVC), polystyrene (PS), styrenecopolymer, polyimide (PI), polypropylene (PP), polytetrafluoroethylene(PTFE), polynorbornene (PN), poly(4-methyl-1-pentene), silicone, andcombinations or co-polymers thereof; silicon; glass; an adhesive, suchas any described herein; as well as combinations thereof (e.g.,combinations of such materials provided in separate layers or within thesame layer). Polymers can include any useful additive, such as, e.g.,fillers (e.g., mica, talc, or calcium carbonate), plasticizers (e.g.,dioctyl phthalate), heat stabilizers (e.g., organo-tin compounds),antioxidants (e.g., phenols or amines), and/or UV stabilizers (e.g.,benzophenones or salicylates). Such materials can be provided in anyuseful form, such as in one or more layers that can be laminated toprovide the assembled apparatus.

Targets and Samples

The present apparatus can be used to detect any useful targets.Exemplary targets include a bacterium, such as Bacillus (e.g., B.anthracis), Enterobacteriaceae (e.g., Salmonella, Escherichia coli,Yersinia pestis, Klebsiella, and Shigella), Yersinia (e.g., Y. pestis orY. enterocolitica), Staphylococcus (e.g., S. aureus), Streptococcus,Gonorrheae, Enterococcus (e.g., E. faecalis), Listeria (e.g., L.monocytogenes), Brucella (e.g., B. abortus, B. melitensis, or B. suis),Vibrio (e.g., V. cholerae), Corynebacterium diphtheria, Pseudomonas(e.g., P. pseudomallei or P. aeruginosa), Burkholderia (e.g., B. malleior B. pseudomallei), Shigella (e.g., S. dysenteriae), Rickettsia (e.g.,R. rickettsii, R. prowazekii, or R. typhi), Francisella tularensis,chlamydia psittaci, Coxiella burnetii, Mycoplasma (e.g., M. mycoides),etc.; allergens, such as peanut dust, mycotoxins, mold spores, orbacterial spores such as Clostridium botulinum and C. perfringens;toxins, such as ricin, mycotoxin, tetrodotoxin, anthrax toxin, botulinumtoxin, staphylococcal entertoxin B, or saxitoxin; a virus, such asAdenoviridae (e.g., adenovirus), Arenaviridae (e.g., Machupo virus),Bunyaviridae (e.g., Hantavirus or Rift Valley fever virus),Coronaviridae, Orthomyxoviridae (e.g., influenza viruses), Filoviridae(e.g., Ebola virus and Marburg virus), Flaviviridae (e.g., Japaneseencephalitis virus and Yellow fever virus), Hepadnaviridae (e.g.,hepatitis B virus), Herpesviridae (e.g., herpes simplex viruses),Papovaviridae (e.g., papilloma viruses), Paramyxoviridae (e.g.,respiratory syncytial virus, measles virus, mumps virus, orparainfluenza virus), Parvoviridae, Picornaviridae (e.g., polioviruses),Poxviridae (e.g., variola viruses), Reoviridae (e.g., rotaviruses),Retroviridae (e.g., human T cell lymphotropic viruses (HTLV) and humanimmunodeficiency viruses (HIV)), Rhabdoviridae (e.g., rabies virus), andTogaviridae (e.g., encephalitis viruses, yellow fever virus, and rubellavirus)); a protozoon, such as Cryptosporidium parvum, Encephalitozoa,Plasmodium, Toxoplasma gondii, Acanthamoeba, Entamoeba histolytica,Giardia lamblia, Trichomonas vaginalis, Leishmania, or Trypanosoma(e.g., T. brucei and T. Cruzi); a helminth, such as cestodes(tapeworms), trematodes (flukes), or nematodes (roundworms, e.g.,Ascaris lumbricoides, Trichuris trichiura, Necator americanus, orAncylostoma duodenale); a parasite (e.g., any protozoa or helminthsdescribed herein); a fungus, such as Aspergilli, Candidae, Coccidioidesimmitis, and Cryptococci; an environmental contaminant; a wateradditive; an agricultural marker; a nucleic acid (e.g.,oligonucleotides, polynucleotides, nucleotides, nucleosides, moleculesof DNA, or molecules of RNA, including a chromosome, a plasmid, a viralgenome, a primer, or a gene); a protein (e.g., a glycoprotein, ametalloprotein, an enzyme, a prion, or an immunoglobulin); a metabolite;a sugar; a lipid; a lipopolysaccharide; a salt; or an ion. Targets alsoinclude food-borne pathogens, such as Salmonella (e.g., SalmonellaTyphimurium), pathogenic E. coli (e.g., O157:H7), Bacillus (e.g., B.cereus), Clostridium botulinum, Listeria monocytogenes, Yersinia (e.g.,Y. enterocolitica), Norovirus (e.g., Norwalk virus), Shigella,Staphylococcus aureus, Toxoplasma gondii, Vibrio (e.g., V. vulnificus,V. cholera, V. parahaemolyticus), Campylobacter jejuni, and Clostridiumperfringens; and weaponized pathogens, such as Bacillus anthracis,Yersinia pestis, Francisella tularensis, Brucella (e.g., B. suis),Burkholderia mallei, Burkholderia pseudomallei, Shigella, Clostridiumbotulinum, Variola (e.g., V. major), Filoviridae (e.g., Ebola virus andMarburg virus), Arenaviridae (e.g., Lassa virus and Machupo virus),Clostridium perfringens, any food-borne pathogen (e.g., Salmonellaspecies, Escherichia coli O157:H7, or Shigella), Chlamydia psittaci,Coxiella burnetii, Staphylococcal aureus, Rickettsia (e.g., R.prowazekii or R. rickettsii), Alphavirus (e.g., Venezuelan equineencephalitis virus, eastern equine encephalitis virus, or western equineencephalitis virus), Vibrio cholerae, Cryptosporidium parvum,Henipavirus (e.g., Nipah virus), Bunyaviridae (e.g., Hantavirus or RiftValley fever virus), Flaviviridae (e.g., Japanese encephalitis virus andYellow fever virus), and Coccidioides spp.

The test sample can include any useful sample, such as a microorganism,a virus, a bacterium, a fungus, a parasite, a helminth, a protozoon, acell, tissue, a fluid, a swab, a biological sample (e.g., blood, serum,plasma, saliva, etc.), an environmental sample, etc.

Reagents

The present apparatus can include any number of useful reagents on-chip.Exemplary reagents include a sterilization agent (e.g., a bleach, suchas sodium hypochlorite or calcium hypochlorite; an oxidizer, such aschlorine dioxide, sodium dichloroisocyanurate, a peroxide, ethyleneoxide, ozone gas, peracetic acid, hypochlorous acid, etc.; a surfactant,such as a cationic, anionic, nonionic, or zwitterionic surfactants, aswell as combinations thereof; an antibiotic; a catalyst; an enzyme; aphage, e.g., a bacteriophage; a disinfectant, such as glutaraldehyde,stabilized hydrogen peroxide, peracetic acid, or formaldehyde; abiocide; an antiseptic; a detergent; a deodorant; and combinationsthereof, where the sterilization agent can be in gas, liquid,semi-solid, or solid form, such as a powder, pellet, granule, gel,lyophilized, or freeze-dried forms), a detection agent (e.g., a dye,such as an electroactive detection agent, a fluorescent dye, aluminescent dye, a chemiluminescent dye, a colorimetric dye, aradioactive agent, etc.; a particle, such as a microparticle, ananoparticle, a latex bead, a colloidal particle, a magnetic particle, afluorescent particle, etc.), a label (e.g., an electroactive label, anelectrocatalytic label, a fluorescent label, a colorimetric label, aquantum dot, a nanoparticle, a microparticle, a barcode, a radio label(e.g., an RF label or barcode), avidin, biotin, a tag, a dye, a marker,an enzyme that can optionally include one or more linking agents and/orone or more dyes), an amplifying agent (e.g., a PCR agent, such as apolymerase, one or more deoxyribonucleotide triphosphates, a divalentmetal (e.g., MgCl₂), a template DNA, a primer (e.g., for binding to aselective region of the target nucleic acid, such those encoding for theprotective antigen and/or capsule of B. anthracis)), a capture agent(e.g., such as a protein that binds to or detects one or more markers(e.g., an antibody or an enzyme), a globulin protein (e.g., bovine serumalbumin), a nanoparticle, a microparticle, a sandwich assay reagent, acatalyst (e.g., that reacts with one or more markers), an enzyme (e.g.,that reacts with one or more markers, such as any described herein)), acell medium (e.g., selective media or other additives to select for orgive advantage to a particular biological target during microculture,including agar, such a nutrient agar, blood agar, heart-infusion agar,where any of these can optionally include 5-7% horse or sheep bloodagar; selective media, such as PLET (polymyxin B-lysozyme-EDTA-thallousacetate) agar for B. anthracis; nutrient media; minimal media;differential media; nutrient broth; or brain-heart infusion broth, whereany of these can optionally include one or more antibiotics to selectfor or against particular targets, one or more nutrients (e.g., a carbonsource, such as glucose), one or more enzymes, one or more host cells,and/or one or more salts), a detergent (e.g., sodium dodecyl sulfate(SDS)), a surfactant (e.g., Tween 20, Triton X-100, glycerin,polyvinylalcohol (PVA), polyvinylpyrrolidone (PVP), or polyethyleneglycol (PEG)), a buffer (e.g., a phosphate or borate buffer), an alcohol(e.g., from about 1% v/v to about 10% v/v methanol, ethanol, orisopropanol), a preservative (e.g., sucrose or trehalose), a blockingagent (e.g., gelatin, casein, bovine serum albumin, IgG, PVP, or PVA), abead (e.g., a glass bead, silica bead, etc., such as to aid in mixing),etc., as well as combinations thereof.

In some embodiments, the device or method includes a stabilizedsterilization agent. For example and without limitation, the stabilizedsterilization agent includes one or more chelators (e.g., chelators forone or more metal ions, which can decompose one or more sterilizationagents, such as an aromatic amine), detergents, surfactants (e.g., anonionic, cationic, or anionic surfactant), alkaline agents (e.g.,urea), preservatives (e.g., ethylenediamine tetra acetic acidcompounds), scavenging agents (e.g., free radical scavenging agents,such diarylamines or substituted dihydroquinolines), sequestrants (e.g.,organic phosphonic acid), stabilizers (e.g., pyrophosphate,pyrophosphoric acid, dipicolinic acid, acetic acid, propionic acid,sulfonates, sulfates, phosphates, organic peroxycarboxylic acid, astannate compound, organic phosphonic acids, amine-substitutedphosphonic acids, alkyleneaminomethylene phosphonic acids, carboxylicacid substituted N-containing heterocyclics, aminopolycarboxylic acids,polyaminocarboxylic acids, tin-based compounds, phosphoric acids,alkylbenzene sulfonates with 6-18 carbon atoms, alkyl sulfates, andwater-soluble salts of these acids), and/or buffers to inhibit or reducedecomposition of the sterilization agent. Exemplary stabilizedsterilization agents include stabilized hydrogen peroxide agents,stabilized peracetic acid, and stabilized hydrogen peroxide-peraceticacid agents, which can optionally include one or more stabilizers (e.g.,any described herein). Additional stabilized sterilization agents aredescribed in U.S. Pat. Nos. 2,347,434; 2,590,856; 2,609,391; 4,051,058;5,656,302; and U.S. Pub. No. 2010/0021558, each of which is incorporatedby reference herein in its entirety.

In other embodiments, the stabilized sterilization agent is provided ina form that increases storage life. Such forms include powder, granule,pellet, gel, lyophilized, freeze-dried, and/or foam forms of thesterilization agent. Such forms can optionally include one or morechelators, detergents, surfactants, alkaline agents, preservatives,scavenging agents, sequestrants, stabilizers, and/or buffers, such asany described herein.

In some embodiments, the device or method includes an electroactive orelectrocatalytic detection agent or label. Such electroactive orelectrocatalytic agents include any compound or chemical capable ofproducing a redox (reduction/oxidation) reaction in which electrons areused or produced, thereby changing the properties of electronflow/transfer (e.g., conduction, resistance, capacitance, etc.), andbeing used for electrochemical detection. Exemplary electroactive orelectrocatalytic detection agents and labels include a redox enzyme(e.g., horseradish peroxidase or glucose oxidase), a mediator chemical(an ‘electron shuttle’), a nanoparticle (e.g., a catalytic nanoparticle,such as those including palladium or palladium-gold that can catalyzethe redox reaction, or a detectable nanoparticle, such as gold orplatinum nanoparticles), a redox active chemical (e.g., ferricyanide,ferrocene, ruthenuim bipyridine, etc.), as well as combinations of anyof these with one or more antibodies or capture agents (e.g., where oneor more catalytic nanoparticles, redox enzymes, or redox activechemicals are directly or indirectly attached (e.g., by a linking agentor a conjugate pair (e.g., biotin-avidin)) to antibodies or othercapture reagents, where such combinations can be detected at anelectrode). In particular embodiments, such electroactive orelectrocatalytic agents are immobilized on one or more electrodes (e.g.,on an electrode array of the device). Exemplary electroactive orelectrocatalytic agents are described in Harper J C et al., “Selectiveimmobilization of DNA and antibody probes on electrode arrays:simultaneous electrochemical detection of DNA and protein on a singleplatform,” Langmuir 2007 Jul. 31; 23(16):8285-7; Polsky R et al.,“Electrically addressable diazonium-functionalized antibodies formultianalyte electrochemical sensor applications,” Biosens. Bioelectron.2008 Jan. 18; 23(6):757-64; and Polsky R et al., Reagentlesselectrochemical immunoassay using electrocatalytic nanoparticle-modifiedantibodies,” Chem. Commun. (Camb.) 2007 Jul. 14; (26):2741-3, each ofwhich is incorporated herein by reference in its entirety.

In some embodiments, the cell medium further includes one or more livingcells. For example, when the target is a virus, then the cell medium caninclude living cells or host cells to allow the virus to infect and/ormultiply. Exemplary cells include a bacterium, a fungus, a mammaliancell, a plant cell, a eukaryote, a prokaryote, etc. In furtherembodiments, the cell medium is a stabilized cell medium (e.g., cellmedium in powder, granule, pellet, gel, lyophilized, freeze-dried,and/or foam forms).

Additional Components

The present apparatus can include one or more additional components, asdescribed herein. For instance, one or more detection components thatallow for detection by electrochemical, colorimetric, fluorescent,western blot, immunohistochemistry, immunoassay (e.g., lateral flowassay), immunochromatography, radio immunoassay, optical immunoassay,enzyme immunoassay, and chemiluminescence, and/orelectrochemiluminescence methods in any useful format. Exemplarydetection components include a capillary bed, e.g., a lateral flow assaystrip having a membrane to bind one or more capture agents, such as anydescribed herein; a transducer, such as an optical sensor (e.g.,including measuring one or more of fluorescence spectroscopy,interferometry, reflectance, chemiluminescence, light scattering,surface plasmon resonance, or refractive index), a piezoelectric sensor(e.g., including one or more quartz crystals or quartz crystalmicrobalance), an electrochemical sensor (e.g., one or more of carbonnanotubes, electrodes, field-effect transistors, etc.), an ion selectiveelectrode, an ion sensitive field effect transistor (e.g., a n-p-n typesensor), a light addressable potentiometric sensor, an amperometricsensor (e.g., having a two-electrode configuration (including referenceand working electrodes) or a three-electrode configuration (includingreference, working, and auxiliary electrodes)), an impedimetric sensor,a disk electrode, a spherical electrode, a plate electrode, ahemispherical electrode, a planar electrode, a three-dimensionalelectrode, a porous electrode, a post electrode, a microelectrode (e.g.,having a critical dimension on the range of 1 to 1000 μm, such as aradium, width, or length from about 1 to 1000 μm), or a nanoelectrode(e.g., having a critical dimension on the range of 1 to 100 nm, such asa radium, width, or length from about 1 to 100 nm), as well as arraysthereof; fiber optics, such as for excitation and collection forfluorescence detection; and/or integrated wave-guides, circular orelliptical microlenses, and/or photodiodes, as well as arrays thereof.

In particular embodiments, the LFA strip includes a sample pad at theproximal end of the strip to receive the sample from an open valve; aconjugate pad downstream of the sample pad and including one or morecapture agents that bind to the target in the sample, thereby forming acomplex, where the capture agent is optionally embedded in a dissolvablematrix; a capillary bed downstream of the conjugate pad and configuredto receive the complex, if present, where the capillary bed is amembrane and includes a test area having one or more specific detectionagents that bind to the complex; and an absorption pad at the distal endfor absorbing excess sample volume. In further embodiments, thecapillary bed includes a control area having non-specific detectionagents that indicate sample flow to the distal end of the strip. In someembodiments, the capture agent is a particle (e.g., a nanoparticle)conjugated to an antibody specific for the target, and the specificdetection agent is an antibody specific for the target. In furtherembodiments, the non-specific detection agent is a control antibody thatnon-specifically binds the complex.

The device can include one or more separation/extraction components(e.g., filters, posts, membranes, weirs (optionally including beads),matrices, or high voltage electrodes for performing on-chip capillaryelectrophoresis separations); heating components (e.g., electrodes orfilaments); pumps (e.g., active or passive pumps, such as a low flowrate peristaltic pump or application of negative pressure, such as byactuating a valve); a membrane (e.g., placed within a channel and/or achamber); a multifunctional sensor (e.g., to measure temperature,strain, and electrophysiological signals, such as by using amplifiedsensor electrodes that incorporate silicon metal oxide semiconductorfield effect transistors (MOSFETs), a feedback resistor, and a sensorelectrode in any useful design, such as a filamentary serpentinedesign); a microscale light-emitting diode (LEDs, such as for opticalcharacterization of the test sample); an active/passive circuit element(e.g., such as transistors, diodes, and resistors); an actuator; awireless power coil; a device for radio frequency (RF) communications(e.g., such as high-frequency inductors, capacitors, oscillators, andantennae); a resistance-based temperature sensor; a photodetector; aphotovoltaic cell; a diode; one or more components to operate atransducer, such as a power source to operate an electrode; adata-processing circuit powered by the power source and electricallyconnected to the transducer (e.g., a counter electrode, a referenceelectrode, and at least one said working electrode); and/or one or morecomponents for autonomous remote monitoring of a sample, such as ananalog-to-digital converter, a radiofrequency module, and/or a telemetryunit (e.g., configured to receive processed data from a data-processingcircuit electrically connected to the detection component and totransmit the data wireles sly).

Kits

The present apparatus can further be provided in a kit. The kit caninclude one or more of the following: a collection swab for collectingthe test sample, a source for actuating the valve(s) (e.g., an externalpermanent magnet), an external heater for incubating the test samplewithin the apparatus, a detection component (e.g., a light-emittingdiode and/or a photodiode), a power source (e.g., when the deviceincludes one or more electrically activatable component, such as anelectromagnet), and/or a telemetry unit (e.g., any described herein).

Methods of Use

The present apparatus includes one or more valves that can be integratedwith any assay for detecting any target of interest. In particularembodiments, the target is a biological agent that can be amplified, andthe apparatus is adapted to be a microculture device, which canoptionally further include a component for detecting the presence of oneor more targets.

In particular embodiments, the apparatus includes a culture chamberon-chip, as well as other structures to maintain or test this culture.For instance, the apparatus can include specific fabrication methodsand/or microfluidic geometries to deliver cell media and nutrients or topromote microcirculation in a bioreactor (e.g., by using negativepressure); integration of cell culture chambers with an array ofelectrodes (e.g., to monitor cell migration); and/or maintenance of cellviability under various controllable conditions for basic studies ofcellular growth/behavior using external devices (e.g., a microscopes) tomonitor cells. Such an apparatus can include a microwell, e.g., asdescribed in U.S. Pat. No. 8,257,964; a perfusion system, e.g., asdescribed in U.S. Pub. No. 2012/0231976; an electrode array, e.g., asdescribed in U.S. Pub. No. 2009/0251155; a sensor, e.g., as described inU.S. Pub. No. 2011/0129850; a module to measure or detect generalmetabolites/byproducts from known cells under well-controlledconditions, e.g., as described in U.S. Pub. No. 2012/0214189; abiodetection or detection components including a capture agent, e.g., ina high-density barcode array form, as described in U.S. Pub. No.2009/0036324, where the barcode array can be used to detect multipleproteins and/or genes from a single cell via on-chip single cellculture, lysis, mRNA, and protein isolation/purification, such as thosein an integrated microfluidic device described in U.S. Pub. No.2009/0053732; methods for controlling channel resistance or flow ratefor use with one or more capture agents, e.g., as described in U.S. Pub.Nos. 2009/0053732 and 2013/0224729; methods for biodetection usingprobes for target DNA, e.g., methods for determining phenotype usingmolecular inversion probes, as described in U.S. Pub. No. 2013/0224729;methods for detecting proteins, e.g., methods for detecting nascentproteins by using primers encoding for N- or C-terminal markers, asdescribed in U.S. Pat. No. 8,278,045 and U.S. Pub. No. 2011/0250609;and/or use of fluorogenic compounds as detecting agents, e.g., use ofFRET-based compounds that are cleavable by one or more enzyme, such asthose described in U.S. Pat. Nos. 6,566,508 and 6,372,895. Furthermore,such apparatuses can be desirable for basic science studies in cellbiology, pharmacology, and immunology. For example, such apparatuses canbe amenable to high-throughput screening of drugs on living cells.

Further exemplary uses and components to effect such uses are providedin U.S. Pat. Nos. 6,566,508, 6,372,895, 8,257,964, and 8,278,045; U.S.Pub. Nos. 2009/0036324, 2009/0053732, 2009/0251155, 2011/0129850,2011/0250609, 2012/0214189, 2012/0231976, and 2013/0224729; Int. Pub.Nos. WO 01/14578, WO 2008/079320, and WO 2009/012340; and EP Pub. Nos.1204671, 1210449, 2108955, 2167634, and 2464753, each of which isincorporated herein by reference in its entirety.

In particular embodiments, the present apparatus include use of a cellculture to amplify the biological target (e.g., not just maintain cellviability or maintain well-controlled culture conditions); and use of anunknown sample and detecting the cells themselves or highly specificmetabolites/byproducts to identify the presence or absence of aparticular cell in the original unknown sample. These characteristicsare fundamentally different than a platform for maintaining cellviability for various biological studies.

EXAMPLES Example 1: Prototype Device

We will now provide fabrication details for prototype devices that weremade and tested. As explained below, the devices were laminated fromsheets of polymethyl methacrylate (PMMA) and biaxially-orientedpolyethylene terephthalate (bo-PET, often marketed under the tradename“Mylar™”). The various perforations were cut out using a carbon dioxidelaser cutter. Lamination was performed under pressure using a hydraulicpress or roller press. The valve elements were pressed onto theiradhesive-coated seats using the hydraulic press.

The adhesive used for the valve seats was an acrylic-based adhesivemarketed by Adhesives Research of Glen Rock, Pa., under the productnumber ARCARE-90445. To achieve a desired amount of adhesion between thevalve element and the valve seat, a controlled amount of pressure wasapplied for a controlled time duration. Further details are providedbelow. It should be noted in this regard that the amount of adhesion isalso dependent on the contact surface area between the adhesive layerand the valve element, and that for that reason, our process parametersshould be adjusted for design variations in which that area is modified.

The valve element was a cylindrical N-52 magnet (composed ofneodymium-iron-boron alloy) 0.1875 inch in diameter and 0.0625 inch inheight. The external magnet used for manual actuation was a cylindricalN-52 magnet 0.375 inch in diameter and 0.375 inch in height.

FIGS. 9A-9B and 10 provide views of the various layers that wereassembled to create the prototypes. FIG. 9A-9B is a perspective,exploded view of a prototype 3000, and FIG. 10 provides plan views ofthe respective layers. Beginning at the top and proceeding downward insequence, the layers were as described below.

The cap assembly 3120 includes an upper (“cap”) portion of 1.5 mm PMMA,a 3.2-mil layer of adhesive, and a lower (“stem”) portion of 1.5 mmPMMA, followed by 3.0-mil coated paper and 31.2-mil adhesive (together3125). The stem portion plugs the fill-hole 3110 in the underlying PMMAsheet.

A structural and legend-bearing assembly 3100, 3200 includes 0.5-mm or0.58-mm PMMA 3101 coated on the bottom side with adhesive, a 3-miltransparency film 3150 of Mylar™ bearing such printed legends as mightbe desired, and 1.08-mm PMMA 3201 coated on the top side with adhesive.The PMMA substrate 3101 includes openings 3150, 3151 for placement ofthe external magnet so that it is aligned with one of the two valves.

The sample chamber (or “culture chamber”) assembly 3300 includes a1.5-mm sheet 3302 of PMMA perforated to define the sample chamber(including a side branch for disposition of its valve magnet after valveopening), preceded and followed by 3.2-mil adhesive layers 3301, 3303.The sample chamber 3310 is formed by openings 3311-3313.

The magnetic valve assembly 3400, 3500 includes an 0.50-mm PMMA sheet3401 followed by an 0.5-mm or 0.58-mm PMMA sheet 3501 coated on the topside with adhesive, both sheets perforated to define the vias 3451,3452, 3461, 3462 and valve seats 3450, 3460 for the respective sampleand sterilization valve magnets 3800, 3810. In one embodiment, the vias3452, 3462 are defined in the lower sheet 3501 and, as explained above,the upper sheet 3401 has slightly larger perforations 3451, 3461 so thatthe valve element will rest on a shoulder portion of the lower sheet3501 and within the larger via in the upper sheet. In anotherembodiment, the upper sheet has a slightly smaller perforation (than thevia in the lower sheet), such that the valve element will rest on ashoulder portion of the upper sheet and within the larger via in thelower sheet.

A sterilization chamber and LFA assembly 3600, 3700 includes a 1.5-mmPMMA sheet 3602 perforated to define the sterilization chamber 3620(openings 3621-3623) and LFA chamber 3610 (openings 3611, 3612) andpreceded and followed by 3.2-mil adhesive layers 3601, 3701, followed bya 1.5-mil PMMA structural base layer 3702. An absorbent sensor strip3660 for use as an LFA capillary bed is placed in the LFA chamber andheld in place by the overlying and underlying adhesive layers.

In an illustrative assembly procedure, the processing temperature wasambient temperature in the range 20-25° C. The processing pressure foradhesive lamination of sheet to sheet was about 3000 psi. The processingpressure for magnet to adhesive was 10-30 psi. The duration forapplication of pressure in each step for joining layers or for adheringa magnet was two minutes. We found that for optimal adhesion, theduration should be carefully controlled, because too little or too muchtime could result in poor adhesion. The area of each adhesive-to-magnetinterface was 30.3 sq. mm.

Example 2: Amplification of Biological Targets via On-Chip Culture forBiosensing

Anthrax poses a significant threat to US National Security asdemonstrated by the 2001 terrorist attacks targeting the US PostalService and Hart Building. The causative agent, Bacillus anthracis, isubiquitous, and more importantly, found in countries harboringterrorists. Between the years 2005-2012, more than three thousand B.anthracis outbreaks were reported. This is likely an underestimation ofthe incidence and prevalence of the disease. Anthrax commonly causessudden death in livestock, and consequently, is routinely isolated andpropagated by indigenous populations to diagnose the disease. Thispractice of isolation and propagation in labs with little-to-no securityin countries harboring terrorists drastically increases laboratories'repositories of B. anthracis, and escalates the risk that the agent canbe stolen for nefarious purposes. In order to mitigate this risk, asimple and inexpensive assay is needed to reduce the amount of B.anthracis handled in the laboratory and eliminate all viable organisms.

Despite significant progress made in the development of biosensortechnologies the utility of many assays remains limited. Commonly, theseassays suffer from the inability to detect the biological target at orbelow the infectious dose. For example, lateral flow assays (LFA) usedfor biodetection permit simple, one-step sample processing without theneed for multiple washing and labeling steps, greatly simplifyingpoint-of-use in the field. However, detection limits for LFAs range from10⁶-10⁷ cells/spores per mL sample. This sensitivity is not low enoughto be practical, where the infectious dose is commonly 10²-10³cells/spores.

The vast majority of biodetection platforms rely on amplification of thebiotarget (e.g., PCR amplification of DNA) or amplification of thesignal (e.g., catalytic labels) to reach relevant detection limits. Thistypically requires devices to be complex, requiring washing and labelingsteps, multiple reagents which may need refrigeration, power, and highskill to operate. These are undesirable attributes, especially inresource limited environments. Amplification of the biological targetprior to downstream biodetection that is ultra-low cost and is verysimple to operate can provide a transformational step in allowingexisting bioassay technologies to detect practical concentrations/levelsof the biological target.

Accordingly, we have developed a portable robust device foramplification of biological targets facilitating subsequent biodetectionthat is ultra-low cost, requires no power or instrumentation to operate,no cold chain (e.g., no refrigeration nor freezing) to maintainefficacy, and can be operated by individuals with little to no technicaltraining. The self-contained credit-card sized device employs on-chipmicroculture methods to amplify the biological analyte prior todownstream detection, improving detection limits by more than fourorders of magnitude (detection from 10² spores/mL initial inoculum of B.anthracis demonstrated using downstream LFA).

Additionally, the device utilizes chemicals and bacteriophage tosterilize the contents following assay. Self-decontamination iscritically important for minimizing potential malicious use of theamplified bacterial sample following assay. This device has thepotential to prevent the currently common practice of isolation andpropagation of biothreat agents by indigenous populations to diagnosedisease in countries harboring terrorists.

This device can also be used for other applications including detectionof food-borne bacteria (e.g., E. coli, Salmonella,etc.) and bacteria ofmedical interest (e.g., Staphylococcus, Streptococcus, Gonorrhoeae,etc.). This device can also detect multiple agents by modifying thedownstream detection method to be multianalyte.

An exemplary device is provided herein. A plastic multilayeredmicrofluidic device was prepared via CO₂ laser machining of plasticlaminates by ablation. Acrylic adhesive coatings on the plastic sheetsprovided a route to multilayer structures via simple hydraulic pressingat room temperature (see FIG. 2A-2B). This approach allowed for rapidand inexpensive development of robust devices. FIG. 11A-11B provides anintegrated on-chip culture and subsequent biodetection device. Thedevice includes a sample port 4110 and cap 4120, which is connected tothe microculture chamber 4310 including media and density lines 4150. Afirst valve (the LFA valve including a valve element 4800 and a via4450) provides fluidic communication between the microculture chamber4310 and the LFA chamber having the LFA capillary bed 4660. The secondvalve (the sterilization valve including a valve element 4810 and a via4460) provides fluidic communication between the microculture chamber4310 and the sterilization chamber 4620.

Fluid moves between chambers on different Z planes (3D) by simpleshaking and/or tapping the device (FIG. 11A), and valves are actuated byuse of an external magnet (FIG. 1B).

As shown in FIG. 11B, the device includes a culture chamber 4310integrated with a lateral flow assay or lateral flow immunoassay (bothreferred to as LFA) 4660 for biodetection. The volume of thismicroculture chamber can be ˜300 mL. The entire device, including themicroculture chamber, remains sealed during the culturing period.Culture can be performed at room temperature, or at elevatedtemperatures, by placing the device in an incubator or in/near anotherheat source. Culturing period is variable depending on the compositionof the sample and the culture matrix, but ranges typically between 24and 72 hours.

As shown in FIG. 1B, the magnet valve is held in place by a thinadhesive ring, preventing access of the microculture solution in theupper chamber to the underlying LFA. An external magnet can be used toremove the magnet from the thin adhesive ring, exposing the verticalconnection between the two chambers. A substance (e.g., liquid or solid)then transfers between chambers by simply shaking the device. Thismagnetic valve configuration is ultra-low-cost (about $0.19 per magnet),requires no power, is simple to operate, and allows assay contents toremain sealed within the device.

Live culture tests were conducted (FIG. 12A). With a 1000 sporeinoculum, the sample was incubated at 37° C. with no agitation. After 24hours, a visibly turbid culture was observed. Optionally, glass beadscan be included in the culture chamber to aid in mixing prior toexposing the sample to the LFA strip. Following the assay, asterilization solution can be used to destroy the culture. FIG. 12Bprovides a test experiment using a commercially available LFA strip,where B. anthracis was detected with 1000 spore inoculum, which wasincubated at 37° C. with no agitation for 24 hours. This device provideda three orders of magnitude improvement in LFA detection limit.

Lower spore counts were also detected. FIG. 13A-13C provides the resultsof experiments with a lower spore count of 100 spore inoculum andincubation at 37° C. Experiments were conducted with no agitation andusing a commercially available LFA strip. The culture time included 24,48, or 72 hours (in FIG. 13A-13C, respectively), and an additionaldevelopment time of about 15 to 60 minutes for the LFA strips shown inFIG. 13A-13C. This device provided a four orders of magnitudeimprovement in LFA detection limit.

Various advantages are observed with this cartridge device. First, thedevice is simple to operate and interpret. Further, manufacturing costsare minimized by the low cost of the magnet actuated valves andprototyping of the device. In addition, the device is completely sealed,which reduces contamination of the assay system, handling of potentiallycaustic sterilization agents (e.g., the sterilization agent can beloaded during assembly, and the device can include no exit ports), anddispersing potentially hazardous biologics after culturing. As shown,the detection limit was improved by employing on-chip culture of thetarget inoculum. To minimize further exposure of anthrax to others, thedevice includes a chamber for subsequent sterilization of the culture.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe claims.

Other embodiments are within the claims.

1. A method for amplifying a target in a sample, the method comprising:introducing the sample within a first reservoir of a first apparatus,wherein the first apparatus comprises a first outlet in fluidiccommunication with the first reservoir, and wherein the first apparatusis configured to allow for in-field or real-time amplification of thetarget; and incubating the sample within the first reservoir, therebyamplifying the target within the sample and providing an amplifiedsample.
 2. The method of claim 1, further comprising sterilizing theamplified sample during or after the incubation step.
 3. The method ofclaim 1, wherein the first apparatus further comprises a first valveelement, and a first valve seat conformed to support the first valveelement in a position that closes the first outlet.
 4. The method ofclaim 3, further comprising opening the first outlet by applying amagnetic field to the first valve element adhesively bonded to the firstvalve seat so as to produce a magnetic force configured to detach thefirst valve element from the first valve seat.
 5. The method of claim 1,further comprising introducing the amplified sample within an assaychamber, wherein the first outlet leads to the assay chamber and theassay chamber is configured to include one or more detection agents foridentifying the target, thereby identifying whether or not the target ispresent within the sample.
 6. The method of claim 5, wherein the assaychamber is provided in the first apparatus or in a second apparatushaving an inlet in fluidic communication with the assay chamber.
 7. Themethod of claim 5, further comprising sterilizing the amplified sampleafter identifying the target.
 8. The method of claim 7, wherein thesterilizing step comprises: opening a second outlet in fluidiccommunication between a second reservoir and the assay chamber, byapplying a magnetic field to a second valve element adhesively bonded toa second valve seat conformed to support the second valve element in aposition that closes the second outlet, wherein the magnetic fieldproduces a magnetic force configured to detach the second valve elementfrom the second valve seat; and introducing a sterilization agent fromthe second reservoir into the assay chamber.
 9. The method of claim 8,wherein the fluidic communication between the second reservoir and theassay chamber occurs through an open passageway in the first reservoir.10. The method of claim 5, wherein the detection agent is one or more ofa dye, a particle, a marker, or a label.
 11. The method of claim 10,further comprising measuring the presence of a detectable signal fromthe detection agent by electrochemical, colorimetric, fluorescent,western blot, immunohistochemistry, immunoassay, immunochromatography,radio immunoassay, optical immunoassay, enzyme immunoassay,chemiluminescence, and/or electrochemiluminescence methods.
 12. Themethod of claim 11, wherein the measuring step comprises detection bylateral flow assay.
 13. The method of claim 11, wherein the measuringstep comprises detection of a plurality of targets.
 14. The method ofclaim 13, wherein the plurality of targets includes one or morefood-borne pathogens.
 15. The method of claim 14, wherein at least onefood-borne pathogen is selected from the group consisting of Salmonella,Escherichia coli, Bacillus cereus, Clostridium botulinum, Listeriamonocytogenes, and Yersinia.
 16. The method of claim 13, wherein theplurality of targets includes one or more weaponized pathogens.
 17. Themethod of claim 16, wherein at least one weaponized pathogen is selectedfrom the group consisting of Bacillus anthracis, Yersinia pestis,Francisella tularensis, Brucella, Burkholderia mallei, Burkholderiapseudomallei, and Shigella.
 18. The method of claim 1, wherein the firstapparatus is the microculture apparatus comprising: a first reservoircomprising at least one first outlet, wherein the first reservoircomprises a cell medium; a second reservoir, wherein the first outlet isconfigured for fluidic communication between the first and secondreservoirs; a first valve element; a first valve seat conformed tosupport the first valve element in a position that closes the firstoutlet; and a first layer of adhesive deposited on at least a portion ofthe first valve seat and configured to releasably bond to a surface ofthe first valve element when seated in the outlet-closed position,wherein the first valve element is responsive to an applied magneticfield of sufficient strength by detaching from the first valve seat andundergoing a displacement that causes the first outlet to open.